Diabetes has been termed the epidemic of the 21st century and over the past 50 years in Western societies has been doubling in incidence every 15 years. It exacts a huge socioeconomic toll because of its devastating microvascular and macrovascular complications and the need of patients to maintain a lifetime daily therapeutic regimen. Diabetes currently affects around two percent of the population and is increasing in incidence. It is a major cause of blindness, kidney disease and premature death, has immense socio-economic impact and places a heavy burden on healthcare facilities worldwide. The childhood-onset form of diabetes, which accounts for 10% of all cases in humans (IDDM, autoimmune or type 1 diabetes) results from an autoimmune process in which T-lymphocytes specifically destroy the insulin-secreting β-cells of the pancreas. This implies that there is one or more β-cell specific target(s) for destructive T-lymphocytes.
Perhaps the single most important advance of the past three decades in diabetes research has been the recognition that autoimmune destruction of β-cells takes months or years to reach completion. Whereas currently the clinical diagnosis of diabetes is almost never made until the destructive process is nearly complete and insulin injections are required to prevent death, intervention before the insulin-producing cells have been irreversibly destroyed can provide a strategy to prevent progression of diabetes and its complications. It is crucial, therefore, to find a means of accurately predicting the onset of autoimmune (type-1) diabetes before the disease has progressed to the clinical stage.
The autoimmune destruction of pancreatic β-cells is mediated mainly by T-lymphocytes of the CD4 and CD8 lineages. Single CD4 and CD8 clones can either accelerate diabetes progression or induce disease after adoptive transfer into genetically susceptible animals. Normally however, both types of cells act in concert, in both regulatory and effector capacities, at all phases of the disease, and the emergence of autoimmune diabetes is generally viewed as the consequence of their dysregulation. Based on the restricted TCR Vα chain repertoire of diabetes-conferring CD4 and CD8 clones in the well-established nonobese diabetic (NOD) mouse model of autoimmune diabetes, the existence of a restricted number of primary targets for T-cell mediated attack appears likely. It is hypothesized that such targets would be β-cell specific, or at least highly expressed in the β-cell relative to the pancreatic α, PP and D-cells.
The majority of diabetic autoantigens that are defined molecularly have been discovered either by a candidate gene approach, or by serological investigations in diabetic humans. Insulin, the 65 kD form of glutamate decarboxylase (GAD) and the insulin granule membrane proteins ICA512 (IA2) and phogrin (IA2β) are major targets of circulating islet cell autoimmunity in man. Other specificities include carboxypeptidase E, ICA69 and sulphated glycolipids. Of these, only insulin appears to be β-cell specific whereas the others are broadly distributed among neuroendocrine tissues such as the brain, pituitary and adrenal medulla. One possible connection between these molecules is their association with the regulated pathway of secretion in the β-cell.
Although B-lymphocytes may play a role in antigen presentation in type 1 diabetes and circulating autoantibodies provide useful pre-clinical markers for diabetic autoimmunity, it is clear that the humoral response per se contributes little to the pathogenesis of the disease. Given that the production of high affinity antibodies is a T-dependent process, it is reasonable to assume that autoreactive diabetogenic T-cells bind to peptides derived from the same molecules recognized by autoantibodies. Consistent with this assumption, insulin, phogrin and GAD have all been shown to be the target of spontaneous T-cell responses in NOD mice.
However the converse is not necessarily true, and attempts to define the cognate antigen of pathogenic T-cell clones in NOD mice that have been selected solely on the basis of islet reactivity have been largely unsuccessful, suggesting that these do not correspond to known serological markers. Thus, much remains to be learned about the specificity and diversity of CD4 and CD8 T-cell responses and their cognate antigens. Such studies in man are impeded by the general inability to access or image the affected organ and the questionable relevance of T-cell responses detected in the peripheral circulation to immunological events occurring within the islet.
Islet-specific CD4 and CD8 T cell clones have been isolated from spleen, lymph nodes or islet infiltrates of pre- or newly diabetic NOD mice and many accelerate disease in naive recipients. A subset of these transfer diabetes susceptibility to NOD scid and NOD rag2(−/−) mice (which have no endogenous T or B cells) thus demonstrating that individual clones with a restricted, if not unique, antigenic specificity can cause disease. The extent to which natural disease is the result of clonotypic or antigen-restricted responses remains unclear. The restricted usage of Vα chains in the TCRs of many of these clones points to the possibility that a limited number of antigenic epitopes and antigens are involved.
Therefore, there exists a substantial and long-felt need for a more accurate means of detecting autoimmune diabetes in its early stages prior to the onset of clinical symptoms and the requirement of insulin therapy. This need would be met by identification of the molecular target(s) of the CD8 T-cell population that infiltrate the pancreatic islet and selectively destroy β-cells while sparing the adjacent endocrine and exocrine tissue. Identification of such a target would make possible assays for the detection of diabetes-related autoimmunity based on: levels of circulating autoantibodies, lymphocyte proliferative responses to the protein and peptides derived from the protein, detection of lymphocytes in the circulation and tissues that react with MHC class I and MHC class II tetramer molecules that incorporate target IGRP peptides, and detection of lymphocytes in the circulation and tissue using ELISPOT assays that incorporate the protein or derived peptides to stimulate reactive cells. Further, the identification of such a protein target would allow the use of the target molecule as a recombinant protein to alter the response of the immune system in a way that is protective rather than destructive. This would involve the use of recombinant protein, chemically or physically modified forms of the protein, peptide sequences derived from the protein, chemically or physically modified and peptide homologues which could be used as a vaccine.